Scintillation assays are commonly used for a variety of analytical purposes. The phenomenon of scintillation arises when a radioactive particle, such as a negative β-particle (an electron) interacts with and excites (raises above its ground state) certain substances. The excited substance emits light as it returns to its ground state. This process is referred to as scintillation; a substance capable of emitting light via the scintillation process is referred to as a scintillant. The flashes of light can be counted easily by a variety of devices. Assays which rely on scintillation have become widespread because of the ease and accuracy with which the radioactivity can be quantitated.
Electrons emitted by individual radioactive negative β-emitting substances are emitted at one or more distinct energies. Because of the distinct energies, electrons emitted by a specific radioactive material have a distinct pathlength in an aqueous environment. (If a material emits electrons at more than one distinct energy, there will be a corresponding pathlength for each energy.) For example, 3H (tritium) emits an electron with a pathlength of approximately 1.5 μm in aqueous solution. (Approximately 90% of the electrons emitted by tritium are absorbed within 1 μm in water, and the maximum range is approximately 8 μm.) 125I emits two electrons with pathlengths of approximately 1 μm and 17.5 μm, respectively. Cook, Drug Discovery Today 1:287 (1996); and Udenfriend et al., Analytical Biochemistry 161:494 (1987).
Scintillation proximity assay (SPA) makes use of the limited pathlength of certain electron-emitters. Hart et al., Molecular Immunology 16:265 (1979); Hart, U.S. Pat. Nos. 4,271,139 and 4,382,074; and Bertoglio-Matte, U.S. Pat. No. 4,568,649. An exemplary SPA is composed of an analyte in solution, plastic beads which scintillate when exposed to electrons, and a specific binding partner (such as an antibody) bound to the beads and specific for the analyte in solution. If the analyte incorporates a radioactive label which emits electrons of relatively short pathlength, such as tritium, the plastic beads will only scintillate when suspended in solution with the radioactive analyte when the analyte is specifically bound by the binding partner and thus localized near the surface of the beads.
One typical application of such an assay is measuring completion of an enzyme-catalyzed reaction. An antibody specific for the product of the reaction and which does not cross-react with the substrate of the reaction is attached to the scintillating material. The substrate is labeled with tritium, and the reaction allowed to proceed. The antibody-coated beads are added to the reaction mixture at a certain time point. The radioactive product will be localized to the surface of the beads, causing scintillation. Substrate molecules, although radioactive, remain unbound and are, on average, too far away from the beads to produce significant scintillation due to the short pathlength of the electron emitted by tritium. Care must be taken to ensure that such background scintillation is kept to an acceptably low level; for example, an extremely high concentration of substrate may lead to unacceptably high background scintillation. Low background levels are accounted for by suitable controls.
SPAs have been developed and exploited for a variety of analytical purposes. SPAs have been used for radioimmunoassays, competition assays, enzyme kinetic assays, studies of ligand/receptor and antigen/antibody interactions, and studies of cellular processes. Cook, Drug Discovery Today 1:287 (1996); Udenfriend et al., Analytical Biochemistry 161:494 (1987); Baxendale et al., Advances in Prostaglandin, Thromboxane, and Leukotriene Research (ed. Sameulsson et al.) 21:303 (New York: Raven Press, 1990); Baker et al., Analytical Biochemistry 239:20 (1996); Nelson, Analytical Biochemistry 165:287 (1987); and Cook, U.S. Pat. No. 5,665,562.
The SPAs described to date all rely on specific binding interactions, such as antibody-antigen interactions, ligand-receptor interactions, biotinylated reagents which bind to streptavidin-coated beads, chelate complex formation of the species of interest, or other interactions which rely on the precise and specific structural complementarity of binding partners. While this gives SPAs high specificity for an analyte of interest, it also requires extra steps in the preparation of reagents and the time and expense of developing a binding partner system specific to the reaction of interest. It also limits its use to those systems where specific binding partners can be found or developed. For example, specific antibodies are needed for antigen-antibody assays, specific receptors are needed for ligand-receptor assays, chelate ligands must be matched to the geometry of the ion with which they form the chelation complex, and, for a biotin-streptavidin assay, analytes must be derivatized with biotin prior to the assay. If no antibodies or receptors are available for detection of a substance, specific derivatization of the analyte with a member of a binding pair such as biotin-streptavidin is required. This can introduce additional complications in the assay. For example, if a binding member is attached to a substrate in order to follow an enzyme-catalyzed reaction, the binding member may interfere with enzyme-substrate binding, rendering the assay inaccurate or useless for determining the reaction progress.
Other SPAs have been developed based on chelation of substrates by a specific chelator. One such assay is based on the preferential binding of linear nucleotides over cyclic nucleotides to yttrium silicate in the presence of zinc sulfate. The binding is described as a complex ion chelation mechanism. (Technical notes to Amersham Life Science Phosphodiesterase [3H]cAMP SPA Enzyme Assay and [3H]cGMP SPA Enzyme Assay, codes TRKQ 7090 and 7100, Amersham International Plc, Buckinghamshire, England.) Again, such an assay system requires specific matching of the chelator to the substrate, based on the spatial requirements of the chelation mechanism, and cannot be generalized.
Thus, it would be useful to develop a system providing the analytical capability of scintillation proximity assays, but eliminating the need to develop a specific binding regime for each analyte. The present invention accomplishes this goal by utilizing distinct molecular properties of an analyte of interest.
The invention provides for rapid, inexpensive and convenient quantitation of an analyte of interest, avoiding the need for separation of reactants and products and obviating the need for preparation of specific antibodies or receptors or the preparation of reagents derivatized with specific binding partners. The invention thus facilitates assays, and can be used for high-throughput screening of chemical libraries.
All references, publications and patents mentioned herein are hereby incorporated by reference herein in their entirety.